The bacterium Bacillus thuringiensis israelensis (Bti) has been used worldwide as an important mosquito control agent for decades (Lacey, 2007). The active ingredient of Bti is a parasporal crystal complex composed of four Cry proteins (Cry4Aa, Cry4Ba, Cry10Aa and Cry11Aa) and two cytolytic proteins (Cyt1Aa and Cyt2Ba) (Porter et al., 1993). Concerns about potential mosquito resistance development to Bti have led to discoveries of other mosquitocidal toxins with high potency. Cry11Ba produced by B.t. jegathesan (Btjeg) is the single most effective toxin against mosquitoes to date. Cry11Ba shares 58% similarity to Cry11Aa and is 7- to 34-fold more toxic to mosquito larvae than the related Cry11Aa (Delécluse et al., 1995).
The resolved structures of Cry proteins show a conservative 3D-topology, suggesting a common mode-of-action. (Boonserm et al., 2005; Boonserm et al., 2006; Galitsky et al., 2001; Grochulski et al., 1995; Li et al., 1991; Morse et al., 2001). Two models regarding the intoxication process of toxins are proposed [reviewed in (Pigott and Ellar, 2007)]. The colloid-osmotic lysis model suggests that proteolytically activated toxins bind cadherin, oligomerize and then bind glycosylphosphatidylinositol (GPI)-anchored aminopeptidase (APN) and GPI-anchored alkaline phosphatase (ALP) to induce toxicity (Bravo et al., 2004). An alternative model proposes the activation of intracellular signaling pathways by toxin monomer binding to cadherin without the need of the toxin oligomerization step to cause cell death (Zhang et al., 2006). Whether toxicity is independent of toxin oligomerization remains arguable, the toxin-receptor interaction has been elucidated in both models as the major determinant of toxin specificity.
APN has long been implicated as a Cry1 toxin binding protein in a number of lepidopteran species [reviewed in (Pigott and Ellar, 2007)]. As a glycoprotein, APN interacts with Cry toxins through either glycan moieties or amino acid residues. For example, Cry1Ac has been shown to bind an N-acetylgalactosamine (GalNAc) moiety on APNs from Manduca sexta (Burton et al., 1999), Heliothis virescens (Luo et al., 1997) and Lymantria dispar (Valaitis et al., 1997). In contrast, Cry1Aa and Cry1Ab are believed to bind APN only in a carbohydrate-independent manner (Masson et al., 1995; Nakanishi et al., 2002). Yaoi et al. (1999) localized a Cry1Aa binding site on Bombyx mori APN to the region between 135Ile and 198Pro. This region contains amino acid residues RXXFPXXDEP conserved among APNs from different species, and thus has been suggested as a common Cry1Aa binding region (Nakanishi et al., 2002; Nakanishi et al., 1999). Recently, a 112-kDa APN (AaeAPN1) in Aedes aegypti has been identified to bind Cry11Aa through the region between 525Arg and 778Leu. (Chen et al., 2009). Unlike the Cry1Aa binding site near the N-terminus, The Cry11Aa binding region was located to the C-terminal region of AaeAPN1. In our previous study, we identified a 106-kDa APN (AgAPN2) as a Cry11Ba binding protein and putative receptor in An. gambiae (Zhang et al., 2008). The 70-kDa partial AgAPN2 expressed in E. coli binds Cry11Ba with high affinity and blocks Cry11Ba toxicity towards mosquito larvae. This APN fragment shows no similarity to the Cry1Aa binding site. Collectively, the data provide evidence that a few primary amino acid sequences on APNs are probably key factor in determining toxin specificities.
To further characterize interactions between Cry11Ba and 70-kDa AgAPN2t we divided the peptide into two fragments of similar size. We showed that one fragment inherited the inhibitory effect of the 70-kDa peptide. By using a combination of in-frame deletions and binding assays, we located a region (336S-P420) on AgAPN2 that is essential for toxin binding and blocking toxicity. Unexpectedly, we also observed an enhancing effect of another fragment (591G-V843) on Cry11Ba toxicity. This is the first report that a non-cadherin fragment of a Cry toxin-binding protein can act as a synergist of Cry toxicity to pest insects (Chen et al., 2007; Park et al., 2009a; Park et al., 2009b).